The objectives of this proposal are to perform structural and mechanistic analyses on two enzymes from glycolysis, rabbit muscle pyruvate kinase and yeast enolase. Both of these enzymes require multiple equivalents of inorganic cations for their function, and the means by which the metal ions promote activity are not fully understood. Crystal structures which were solved for both of these enzymes during the past budget period, together with structural information obtained from EPR spectroscopic studies, provide the background for the proposed experiments. Clones of genes for both enzymes are available, and site specific mutagenesis can be used to augment the structural studies. For pyruvate kinase, we have crystals for X-ray structural analysis of the fully liganded form of the enzyme-including a form with authentic ATP bound in the active site. The present X-ray structure suggests a plausible mechanism for the monovalent cation activation of the enzyme, and this mechanism will be tested by site specific mutants of residues in the vicinity of the monovalent cation site. Candidates for the general acid/base catalyst, revealed in the structure, will be screened by mutations and structure/function analyses of the mutant enzymes. For enolase, our structure for a new, high pH, low salt, crystal form of the enzyme with two Mg ions and an intermediate state analog bound in the active site indicates that there are stereochemical inconsistencies in the current model of enolase catalysis. We will approach this issue by obtaining crystal structures of bis metal ion complexes of the -enzyme-substrate mixture and of a nitro analog of the proposed carbanion intermediate in the reaction. These studies will be augmented by mutations of specific residues believed to be essential in the two alternative mechanisms and by systematic analyses of the effects of these mutations. The functional importance of the closure of an intricate active site flap will be examined by compromising flap closure through mutagenesis.